Magnetic nanoparticles typically comprise nanocrystals made of oxides (or to a lesser extent of sulfides) of the elements in the forth row of the periodic table (i.e. Cr, Mn, Fe, Co, and Ni). The ability to produce such magnetic nanoparticles is inevitable not only for the general understanding of magnetic properties in a nanometer regime, but also for manifold technical applications ranging from magnetic resonance imaging, drug delivery, catalysts, and biosensing to nanoelectronics, semiconductor materials, and magnetic storage media (reviewed in Lu, A. et al. (2007) Angew. Chem. Int. Ed. 46, 1222-1224).
Magnetic nanoparticles can be synthesized chemically through precipitation of the crystals from basic aqueous solutions. However, the production of particularly dimensioned (“tailored”) nanocrystals via these synthesis routes is significantly hampered by the broad size distribution of the crystal populations obtained. More recently, the synthesis of nanocrystals has been directed to non-aqueous approaches generally resulting in the formation of crystals having not only an improved overall quality but also a narrower size distribution. Nevertheless, in most chemical syntheses reported so far only sub-gram to low gram quantities of monodisperse nanocrystals were obtained, not sufficient for many applications. Furthermore, only a fraction of such synthetic particles constitutes monocrystalline particles having defined magnetic properties. Typically, however, the resulting particles were too small for clinically relevant applications such as magneto-hyperthermic treatment of tumors (Park, J. et al. (2004) Nature Materials 3, 891-895).
Alternatively, biogenic magnetic nanoparticles can be employed that are produced by magnetotactic organisms, predominantly magnetotactic bacteria. The ability of magnetotactic bacteria to orient in the Earth's magnetic field is based on the presence of specific organelles, the magnetosomes, which are membrane-enveloped monocrystalline crystals of a magnetic mineral that are arranged in chain-like structures within the cell. Magnetosomes display a variety of species-specific shapes within the single magnetic domain size range (reviewed in Bazylinski, D. A. and Frankel, R. B. (2004) Nature Rev. Microbiol. 2, 217-230). In the prototypical Magnetospirillum, cubo-octahedral nanocrystals of the mineral magnetite (Fe3O4) having a maximal diameter of 50 nm are synthesized within magnetosome membrane (MM) vesicles. The MM is a phospholipid bilayer of a distinctive biochemical composition. Their specific structural and magnetic properties make these bacterial magnetosomes highly attractive for various nanotechnological and biomedical applications.
However, the biotechnological usability is hampered by the fact that they can only be produced in comparably small amounts due to the methodological difficulties to cultivate magnetotactic bacteria due to fastidious growth requirements. Typically, the cell yields obtained are only about 1 g (fresh weight) per liter of culture, not enough for the large scale production of magnetosomes (Heyen, U. and Schüler, D. (2003) Appl. Microbiol. Biotechnol. 61, 536-544). In addition, genetic interference in or manipulation of the genetic pathway for magnetosome production is cumbersome due to the limited availability of molecular tools for the recalcitrant native host cells.
The biogenesis of functional magnetosomes is highly complex and involves the invagination of magnetosome vesicles from the cytoplasmic membrane, the magnetosomal uptake of iron and the crystallization of magnetite particles, as well as their assembly into chains along a dedicated cytoskeletal structure (Komeili, A. et al. (2006) Science 311, 242-245; Katzmann, E. et al. (2010) Mol. Microbiol. 77, 208-224). Recently, in M. gryphiswaldense, genes controlling magnetosome synthesis within several clusters of a larger (115 kb) genomic magnetosome island (MAI) were discovered, which are interspersed by transposases and genes of unknown function (Jogler, C. et al. (2009) Environ. Microbiol. 11, 1267-1277; Jogler, C. et al. (2011) Proc. Natl. Acad. Sci. USA 108, 1134-1139). Whereas the smaller mamGFDC (2.1 kb), mms6 (3.6 kb) and mamXY operons (5.1 kb) have accessory functions in biomineralization of properly sized and shaped crystals, the large mamAB operon (16.4 kb) encodes proteins essential for iron transport, magnetosome membrane formation, and crystallization of magnetosome particles as well as their assembly and intracellular positioning. However, due to the structural complexity of the magnetosome organelle, current knowledge about particular gene functions is still quite limited and the presumably complex interplay between the numerous factors involved in the production of magnetosomes needs still to be unraveled in closer detail which is further complicated by the limited methodology for genetic manipulation of magnetotactic bacteria that is presently available.
Thus, there is a need for molecular tools as well as corresponding methods in order to improve the yield of biogenic magnetic nanoparticles that can be obtained from bacterial cultures—both in homologous and heterologous genetic backgrounds (i.e. settings). In particular, there remains an ongoing need for techniques that allow for the large-scale production of magnetic nanoparticles in an easy-to-do and cost-efficient manner.
Accordingly, it is an object of the present invention to provide such molecular tools and corresponding methods.